Construction and use of genes encoding pathogenic epitopes for treatment of autoimmune disease

ABSTRACT

The present invention relates to the application of genetic engineering to provide a treatment of autoimmune disease. This is achieved preferably through the introduction of one or more recombinant genes encoding self antigens which are the target of an autoimmune response. In particular the invention provides a method of designing and constructing a gene encoding an encephalitogenic epitope of proteolipid protein, and to the in vivo expression of the gene product by a recombinant retroviral vector. The expression and secretion of the encephalitogenic epitope ameliorates the histopathological and clinical characteristics of experimental autoimmune encephalomyelitis (EAE) in the mouse model for multiple sclerosis (MS).

FIELD OF THE INVENTION

[0001] This invention relates generally to the field of immunotherapyand to the preparation and use of engineered cells having the ability torestore tolerance to self antigens in patients suffering from autoimmunedisease. More particularly, this invention relates to the design andconstruction of a gene encoding an encephalitogenic epitope ofproteolipid protein (PLP), to methods of in vitro and in vivo expressionof a PLP epitope, to methods of in vivo secretion of a PLP epitope, andto methods of transferring the partial PLP gene to a host to amelioratethe progression of an immune response to self antigens derived frommyelin proteins.

BACKGROUND OF THE INVENTION

[0002] The immune system can respond in two ways when exposed to anantigen. A positive response leads to differentiation of T and B cells,antibody production and to immunologic memory. A negative response leadsto suppression or inactivation of specific lymphocytes and to tolerance.Tolerance can be defined as the failure of an organism to mount animmune response against a specific antigen. Normally, an organism istolerant of its own antigens.

[0003] Autoimmune diseases are thought to result from an uncontrolledimmune response directed against self antigens. In patients withmultiple sclerosis (MS), for example, there is evidence that this attackis against the white matter of the central nervous system and moreparticularly to white matter proteins. Ultimately, the myelin sheathsurrounding the axons is destroyed. This can result in paralysis,sensory deficits and visual problems. MS is characterized by a T celland macrophage infiltrate in the brain. Autoreactive myelin-specific Tcells have been isolated from MS patients, although T cells of the samespecificity have been detected in normal individuals. J. M. LaSalle etal., J. Immunol. 147:774-780 (1991), J. M. LaSalle et al., J. Exp. Med.176:177-186 (1992), J. Correale et al., Neurology 45:1370-1378 (1995).Presently, the myelin proteins thought to be the target of an immuneresponse in MS include myelin basic protein (MBP), proteolipid protein(PLP), and myelin-oligodendrocyte glycoprotein (MOG). Individuals who donot mount an autoimmune response to self proteins are thought to havecontrol over these responses and are believed to be “tolerant” of selfantigens. The evidence, therefore, that MS is caused by pathogenic Tcells is necessarily indirect, but the close resemblance which thecharacteristics of this disease bear to those of the murine model,experimental autoimmune encephalomyelitis (EAE), suggest that MS isindeed caused by an aberrant immune response mediated by T cells.

[0004] The RAE mouse model for MS, the subject of intense and fruitfulstudy for several years, displays many of the same histopathological andclinical characteristics as the relapsing remitting forms of MS. The TLymphocyte in Experimental Allergic Encephalomyelitis, Ann. Rev.Immunol. 8:579-621 (1990). EAE can be induced in SJL mice by injectionof mouse spinal cord homogenate (MSCH), MBP, PLP, by the injection ofsynthetic peptides whose sequences correspond to the majorencephalitogenic epitopes of myelin basic protein, MBP 84-104,proteolipid protein, PLP 139-151, or by adoptive transfer of activatedCD4⁺ T_(H1,) but not T_(H2) cells specific for encephalitogenicepitopes. The major encephalitogenic epitopes of myelin-derivedsequences in EAE, such as MBP, can also activate human T cells ofseveral different haplotypes including HLA-DR2. R. Martin, et al., J.Exp. Med. 173:19-24 (1992). The experimental disease is characterized bya relapsing-remitting course (R-EAE) of neurological dysfunction,perivascular mononuclear infiltration and demyelination. CNS damage isprobably mediated by inflammatory cytokines which can activateadditional monocytes and macrophages non-specifically. J. E. Blalock,The Immune System, Our Sixth Sense, The Immunologist, 2:8-15 (1994).

[0005] Although the initial attack in EAE can be induced by theadministration of either T cells specific for MBP or for PLP, closeexamination of reactivities of T cells in the primary and subsequentrelapses demonstrates the presence of T cells which interact withspecificities other than the inducing epitopes. This expansion ofencephalitogenic epitopes is termed “determinant spreading”. S. D.Miller and W. J. Karpus, Immunology Today 15:356-361 (1994), P. V.Lehman, T. Forsthuber, A. Miller, and E. E. Sercarz, Nature 358-155-157(1992), H. Jiang, S. I. Zhang and B. Pernis, Science 256:1213-1215(1992). Antigen specific treatment would therefore, be expected to bemore effective when administered early in the course of the disease,before the onset of increasing epitope complexity and eventualnon-specific inflammation.

[0006] The goal of immunologic therapy is to restore tolerance withoutsuppressing the entire immune system which can lead to complicationssuch as infection, hemorrhage, and cancer. Drugs currently used to treatautoimmune diseases are non-specific immunosuppressive agents, such asanti-inflammatory agents or drugs which can block cell proliferation ordepress proinflammatory cytokines. In general, these agents areeffective for limited duration and subject to devastating complications.

[0007] It is desirable to suppress the immune system in a more specificway to control the response to self-antigens and theoretically “cure”the disease without down-regulating the entire immune system. Severalspecific immunotherapies have been hypothesized and tested in recentyears, many of which are impractical or do not work in humans. Forexample, high affinity peptides can be synthesized which interact withMHC class II molecules and prevent the binding of encephalitogenicpeptides, thereby preventing the activation of pathogenic T cells. A.Franco et al., The Immunologist 2:97-102 (1994). This approach isdisadvantageous in that it is difficult to obtain effectiveconcentrations of inhibitor peptides in vivo. G. Y. Ishioka et al., J.Immunol. 152:4310-4319. In an alternate strategy, peptides which areanalogs of encephalitogenic sequences have been shown to antagonize theT cell receptors of antigen-specific T cells, rendering them unreactive,although the exact mechanism is at present unknown. S. C. Jameson etal., J. Exp. Med 177:1541-1550 (1993), N. Karin et al, J. Exp. Med.180:2227-2237 (1994), V. K. Kuchroo et al, J. Immunol. 153:3326-3336(1994). Oral administration of myelin has been tested and found toinduce a state of immunological unresponsiveness thought to be mediatedby the induction of suppressor T cell or of anergy H. L. Weiner et al.,Annu. Rev. Immunol. 12:809-837 (1994), C. C. Whitacre et al., J.Immunol. 147:2155-2163 (1991), S. J. Khoury et al., J. Exp. Med.0.176:1355-1364 (1992). This treatment has been found to be efficaciousfor some but not all individuals. H. L. Weiner et al., Science259:1321-1324 (1993). Thus, it is evident that improvements are neededto treat MS and other autoimmune disorders with an effective,immunospecific approach.

SUMMARY OF THE INVENTION

[0008] The present invention addresses the disadvantages present in theprior art. In general, the invention is based on the discovery thatrecombinant DNA technology and cell transfer may be employed to restoretolerance to one's own tissues. The present invention provides a meansof preparing and constructing a gene, that when expressed and secretedin vivo, can provide a means of halting the progression of an autoimmunedisease. In further aspects the invention provides a method to constructa gene encoding a portion of a CNS protein, insert the gene sequenceinto a vector and transfect a cell line. In further aspects, theinvention provides a method to construct a gene encoding a portion of aCNS protein, insert the sequence into a retroviral vector, and transducea producer fibroblast cell line to generate supernatant containing therecombinant retrovirus. Histocompatible fibroblasts are transduced withthe recombinant retrovirus encoding a portion of the CNS protein and aredelivered to animals. These fibroblasts continuously secrete a CNSantigen in vivo but do not themselves produce viral particles.

[0009] In accordance with the present invention, we have used syntheticoligonucleotides to construct a gene encoding a portion of the PLPprotein, performed expression of the DNA in combination with variousexpression vectors, and thereby evaluated expression levels of the geneproduct in vitro and in vivo. After transduced histocompatiblefibroblasts that secrete the partial PLP protein are transplanted intoEAE mice, the disease disappears. The effect is the amelioration of bothclinical symptoms and signs and pathological findings.

[0010] In a preferred embodiment of the invention, the producer linePA317 is transduced with the PLP retroviral vector to generatesupernatant containing the recombinant retrovirus. The producer cellline PA317 was developed by Dr. A. Dusty Miller and has been extensivelycharacterized and approved for human use by the FDA for other clinicaltrials, such as for genetic diseases and cancer. Miller and Baltimore,Mol. Cell Biol. 6:2895-2902 (1986), W. F. Anderson, Science 256:808-813.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a map of the partial PLP gene showing the sequence ofthe gene product and restriction sites.

[0012]FIG. 2 is a map of the GlXSvNa vector illustrating restrictionsites and functional features. FIG. 2b illustrates, the entire DNAsequence of GlXSvNa.

[0013]FIG. 3 outlines the method of constructing a GlXSvNa vectorcontaining the PLP gene insert.

[0014]FIG. 4 shows the level of mRNA expressed in transfected andtransduced SJL fibroblast cells as detected by reverse transcriptasePCR. Lane 1 is molecular weight standards, Lane 2 is Negative controlfrom mock transfection, Lane 3 is positive control-PLP-gene plasmid,Lane 4 is cDNA from PLP-transfected SJL fibroblasts, Lane 5 is cDNA fromPLP transduced SJL fibroblasts.

[0015]FIG. 5 demonstrates the level of PLP protein in the supernatantsof transduced fibroblasts as detected by ELISA.

[0016]FIG. 6 demonstrates the level of B-Gal expression in transducedfibroblasts.

[0017]FIG. 7 illustrates the clinical scoring system for chronic EAE.

[0018]FIG. 8 illustrates the histological scoring system for EAE.

[0019]FIG. 9 illustrates the clinical assessment of EAE mice treatedwith retrovirus transduced fibroblasts.

[0020]FIG. 10a shows the pathologic assessment of brain and spinal cordof SJL mice treated with retrovirus transduced fibroblasts, and 10 b isa summary of the pathologic assessment of brain and spinal cord fromDays 55-60 through days 90-95.,

[0021]FIG. 11 shows the histology of SJL mice with chronic EAE treatedwith retrovirus transduced fibroblasts.

[0022]FIG. 12 illustrates the results of proliferation assays using FAEmice treated with PLP-expressing fibroblasts.

[0023]FIG. 13 illustrates the results of proliferation assays with andwithout IL-2 using EAE mice treated with PLP-expressing fibroblasts.

DETAILED DESCRIPTION OF THE INVENTION

[0024] As indicated above, the present invention relates to the use ofengineered cells to restore tolerance to self antigens in patientssuffering from autoimmune disease. The engineered cells can be anymammalian cell. As used herein, the term “engineered” is intended torefer to a cell into which one or more recombinant genes, such as a geneencoding an epitope of a self antigen, has been introduced.

[0025] A gene is a deoxyribonucleotide sequence coding for an amino acidsequence. Recombinantly introduced genes will either be in the form of asynthetic oligonucleotide, a cDNA gene (i.e. they will not containintrons), a copy of a genomic gene sequence, or a hybrid, gene which isa fusion of two or more gene sequences. Optionally the gene may belinked to one or more nucleotide sequence capable of directingexpression of the gene product. Sequence elements capable of effectingexpression of a gene or gene product include but are not limited topromoters, enhancer elements, transcription termination signals,polyadenylation sites, a Kozak box sequence to ensure efficienttranslation, and leader sequences. Optionally, the gene sequence caninclude restriction sites to enable the insertion of additional genesequences. Preferably, the gene will contain a leader sequence to ensurethe gene product is synthesized in the endoplasmic reticulum for laterconstitutive secretion.

[0026] Recombinantly introduced genes carried by the engineered cellscan encode one or more epitope, fragment, domain or mini-protein portionof a protein antigen. Examples of suitable proteins from which anepitope, fragment, domain, or mini-protein may be derived include butare not limited to myelin proteins, acetylcholine receptor, TSHreceptor, and collagen.

[0027] It is believed that protein self-antigens which are the target ofan autoimmune response are highly conserved both among and betweenspecies. Thus, although the invention will primarily be used to treathumans it can also be used to treat animals. Examples of T cell mediatedautoimmune diseases that may be treated using the invention include butare not limited to multiple sclerosis, myasthenia gravis, systemic lupuserythematosus, psoriasis, juvenile onset diabetes, rheumatoid arthritis,thyroid disease and chronic inflammatory demyelinating polyneuropathy(CIDP).

[0028] Expression vectors are generally deoxyribonucleotide moleculesengineered for controlled expression of one or more desired genes. Thevectors may comprise one or more nucleotide sequences operably linked toa gene to control expression of the desired gene or genes. There are anabundance of expression vectors available and one skilled in the artcould easily select an appropriate vector. In addition, standardlaboratory manuals on genetic engineering provide recombinant DNAmethods and methods for making and using expression vectors. Optionally,the vector may encode a selectable marker, for example, antibioticresistance.

[0029] The gene can be inserted into the mammalian cell using any genetransfer procedure. Examples of such procedures include but are notlimited to, RNA viral mediated gene transfer such as retroviraltransduction, DNA viral mediated gene transfer, electroporation, calciumphosphate mediated transfection, microinjection or liposome mediatedgene transfer. The type of procedure required to achieve an engineeredcell that secretes the desired gene product will depend on the natureand properties of the cell. The specific technology for introducing suchgenes into such cells is generally known and well within the skill ofthe art.

[0030] The examples which follow illustrate the design and constructionof a portion of the PLP gene, in vitro and in vivo expression of the PLPgene product, and the in vivo effects of the PLP gene product. Thefollowing examples are presented to illustrate the invention, and arenot intended to limit the scope thereof.

EXAMPLE 1

[0031] Design and Construction of the PLP Gene

[0032] In SJL/J mice, the encephalitogenic epitope of PLP comprisesamino acids 139-151. N Takahashi et al., Cell 42:139-148 (1985), K Sakaiet al., J. Neuroimmunol. 19:21-32 (1988), D. H. Kono et al., J. Exp.Med. 168:213-227. The vector in the present invention is designed inorder that the gene product encoded by it be constitutively secretedfrom fibroblasts. Since the complete PLP protein is a hydrophobictransmembrane protein (H-J. Diehl, M. Schaich, R-M. Buszinski and W.Stoffel, PNAS U.S.A. 83:9807-9811 (1986)), with the encephalitogenicepitope being extracellular, a plasmid encoding amino acids 101-157 andadditional amino acids required for secretion was constructed. Thissequence is hydrophilic in character.

[0033] 1. Oligonucleotide Synthesis and Construction of the PLP pRc/CMVVector

[0034] Oligonucleotides can be synthesized manually, e.g., by thephospho-tri-ester method, as disclosed, for example in R. L. Letsinger,et. al., J. Am. Chem. Soc. 98:3655 (1967), the disclosure of which isincorporated by reference. Other methods are well known in the art. Seealso Matteucci and Caruthers, J. Am. Chem. Soc. 103:3185 (1981), thedisclosure of which is incorporated by reference.

[0035] Preferably, however, the desired gene sequence can be made byautomated synthesis of individual oligonucleotides at 0.2 μMconcentrations. For PLP amino acids 101-157, DNA syntheses wereperformed on a Perkin Elmer/Applied Biosystems Division Model 394 DNAsynthesizer using cyanoethyl-protected phosphoramidites. Thedimethoxytrityl (DMT) group was not removed from the 5′hydroxyl group toallow for purification. After normal cleavage from the resin usingconcentrated ammonium hydroxide and deprotection at 55° C. for 16 hours,the oligonucleotides were purified using oligonucleotide purificationcartridges (CPC) according to the manufacturer's instructions (AppliedBiosystems Inc.) Five oligonucleotides of the following sequences weresynthesized: OLG15′-CGGCGACTACAAGACCACCATCTGCGGCAAGGGCCTGAGCGCAACGGTAACAGGGGGCCAGAAGGGGAGGGGTTCCAGAGGCCAACATCAaGCTCATTCTCTCGAGC-3′, OLG25′-GAGCTTGATGTTGGCCTCTGGAACCCCTCCCCTTCTGGCCCCCTGTTACCGTTGCGCTCAGGCCCTTGCCGCAGATGGTGGTCTTGTAGTCGCCGGGCC-3′, OLG35′-GGGTGTGTCATTGTTTGGGAAAATGGCTAGGACATCCCGACAAGTTTGTGGGCATCACCTATGCTACCCTTAAGTAGGATCCTTCAATAGGTA-3′, OLG45′-AGCTTACCTATTCAAGGATCCTACTTAAGGCTAGCATAGGTGATGCCCA-3′, and OLG55′-CAAACTTGTCGGGATGTCCTAGCCATTTTCCCAAACAATGACACACCCGCTCGAGAGAAT-3′.

[0036] Each purified oligonucleotide was dried under vacuum, washed with1 ml of sterile double distilled water and then concentrated to drynessunder vacuum (Speed vac evaporator; Savant Inc.). 80 pm of each oligomerwas kinased at 37° C. for 1 hour by resuspending in 56.6 μl of 1× kinasebuffer (Polynucleotide Kinase Buffer; Boehringer Mannheim, Indianapolis,Ind.) containing 10 units, of polynucleotide kinase (BoehringerMannheim) and 100 μM of ATP. The individual oligonucleotides werecombined in the presence of 2×SSC (0.03M Sodium Citratei pH 7.0, and0.3M NaCl) in a PCR tube with their respective complementary oligomerpartners for annealing. Each annealed set measured 20011 in volume.Oligomer OLG1 was annealed with OLG2, and oligomers OLG4 and OLG5 wereannealed with OLG3. Annealing was performed in a Perkin-Elmer 9600Thermocycler, programmed as follows: 1)99.9° for 2 minutes, and 2) 99.9°to 40 in 15 minutes. During the temperature descent to 4° C., when thethermocycler temperature reached 37° C., the solution containing theoligomer duplex OLG1 and OLG2 was combined with the solution containingthe oligomers OLG3, OLG4, and OLG5. The descent cycle was then continueduntil it reached 22° C. Subsequently, 5 units (5 μl) of T4 ligase(Boehringer Mannheim, Indianapolis, Ind.) and 45 μl of manufacturer's10×T4 DNA ligation buffer (Boehinger Mannheim, Indianapolis, Ind.) wasadded, and ligation proceeded overnight at 10° C.

[0037] The ligated DNA was precipitated with 2 volumes of 100% ethanoland incubated at −70° C. for 1 hour. The precipitate was centrifuged for30 minutes at 17000×g at 4° C. The supernatant was discarded and pelletwas washed with 1 ml of 70° ethanol and centrifuged for 10 minutes at17000×g at 4° C. The DNA pellet was dried under vacuum (Speed vacevaporator; Savant Inc.) and resuspended in 45 μl sterile doubledistilled water.

[0038] DNA of the correct molecular weight was isolated byelectrophoresis. 5 μl of 10× loading buffer (6.25 g Ficoll and 0.93 gDisodium EDTA/25 ml 10% SDS, Orange G, Xylene Cyanole, and BromophenolBlue) was added to the sample and loaded onto a 14.5 cm×16 cm×0.15 mmurea/acrylamide gel (7M urea/8% acrylamide with 1.1% Bis). TBE (89 mMTris, 89 mM Boric acid, and 2 mM EDTA pH8.0) was used as both gel andelectrophoresis buffer. The sample was electrophoresed at 35 mA untilthe Orange G dye line had migrated within 1 cm of the bottom of the gel.The acrylamide gel was washed twice with water for 5 minutes. After thelast wash, the gel was incubated for 3 minutes in a 500 ml solutioncontaining 10 ul of 10 mg/ml of ethidium bromide, and visualized under aUV-light source. The band corresponding to the ligated DNA was excisedand cut into small pieces for electroelution in an IBI electroelutorapparatus (Model UEA: International Biotechnologies Inc., New Haven,Conn.).

[0039] For electroelution, the salt trap of the apparatus was filledwith 125 μl of 7M sodium acetate/bromophenol blue dye solution. Thebuffer chamber was filled with {fraction (1/2)}×TBE. The sample waselectroeluted for 1 hour at 85V. After removing the eluted DNA, thesample well was washed with {fraction (1/2)}×TBE and combined with theinitial eluate. The eluted DNA was then precipitated overnight at −70°C. with 2 volumes of 100% ethanol. The precipitate was pelleted, washedas previously described, and resuspended in 15 ul of sterile doubledistilled water.

[0040] Preceding the ligation of the eluted partial PLP gene to thepRc/CMV vector (Invitrogen, San Diego, Calif.), the pRc/CMV vectorconstruct was cut with the restriction endonucleases Apa I and Hind IIIaccording to the Manufacturer's instructions (Boehringer Mannheim,Indianapolis, Ind.). The resuspended PLP gene construct was then addedto a 5 μl mixture containing 0.3 kg of pRc/CMV cut vector (2 μl), 1 unitT4 ligase (1 μl) (Boehringer Mannheim, Indianapolis, Ind.), and 2 μl ofManufacturer's 10×T4 DNA ligation buffer (Boehringer Mannheim,Indianapolis, Ind.). The ligated vector was then transformed into thecompetent cell line AG1.

[0041] Transformation proceeded by combining the ligation mixture withthe AG1 cells and incubating it on ice for 20 minutes. The cell/vectormixture was then incubated at 42 for 2 minutes and plated overnight ontoa Luria Broth agar (LB; Bio101, Vista, Calif.) plate, supplemented with80 mg/ml of ampicillin (Sigma, St. Louis Mo.) Colonies were screened forthe correct sequence vector by first isolating the plasmid DNA and thensequencing the DNA.

[0042] To isolate the plasmid, a commercially available plasmidpurification kit, Wizard Minipreps (Promega, Madison, Wis.) was used.Colonies were picked from the LB/Amp plates and grown for 3.5 hours in 5ml of LB medium (BIO 101, Vista, Calif.) supplemented with 80 mg/ml ofampicillin (Sigma, St. Louis, Mo.). 3 ml of the medium was centrifugedat 17000×g at room temperature, for 1 minute to pellet the cells.Isolation of the plasmid proceeded according to the Manufacturer'sinstructions. 1 μg of the isolated DNA was used, for sequencing.

[0043] The oligonucleotide sequence can be checked by methods well knownin the art, such as that described by Sanger et al., PNAS U.S.A. 70:1209(1973) or by the Maxam-Gilbert method, Meth. Enzymology, 65:499 (1977),the disclosures of both of which are incorporated herein by reference.Preferably, the plasmid can be sequenced using an automated DNAsequencer. For the PLP pRc/CMV construct, the plasmid was sequencedusing automated fluorescent DNA sequencing procedures (PerkinElmer/Applied Biosystems Inc, Foster City, Calif.) using the followingprimers: GATTTAGGTGACACTATAG and TAATACGACTCACTATAGGG. These primersprimed off the vector, which flanked the Kozak and “stop” site of thetotal construct. FIG. 1 shows a map of the partial PLP gene showing thesequence of the gene product and restriction sites. At the 5′ end of theconstruct we had previously inserted a hydrophobic leader sequence fromthe MHC class I L^(d) gene to enable the gene product to be synthesizedin the endoplasmic reticulum IER) for later constitutive secretion.Linsk et al. J. Exp. Med. 164:794-813 (1996). In addition, a lysinecodon at the 3′ end was added to ensure that the protein could not beretained in membrane. A Kozak box was included in the construct toensure efficient translation. Restriction sites Afl II and BamHI wereincluded in the construct to allow for insertion of further epitopes.

EXAMPLE 2

[0044] In Vitro Expression of the PLP Protein

[0045] The following experiments were performed in order to demonstratethat the PLP vector encodes a protein which is constitutively secreted.Specifically, the mRNA levels of PLP were evaluated in SJL fibroblastcells transfected with the pRc/CMV-PLP vector, and mRNA and proteinlevels of PLP were evaluated in SJL fibroblast cells transfected withthe pGlPLPSvNa vector.

[0046] 1. Establishment of Fibroblast Cultures

[0047] Syngeneic fibroblasts (derived from SJL mice) were obtained fromDr. G. Dveskler (Uniformed Services University, Bethesda, Md.) andexpanded at 37° incubation using DMEM growth medium, supplemented with5% glutamine and 10% FCS. The cells were harvested and frozen at 1×10⁷cells per vial, and aliquots were quality control tested for mycoplasma,sterility and viability.

[0048] 2. Retroviral Constructs

[0049] A recombinant retroviral vector in which exogenous genes areinserted into a retroviral vector was constructed. The cloning strategywas to construct a pGlXSvNa vector (W. French Anderson, University ofSouthern California) containing the PLP insert from pRc/CMV-PLP. ThepGlXSvNa vector, like most retroviral vectors used in preclinical andclinical trials, is derived from the Moloney murine leukemia retrovirus(Mo-MLV). Rosenberg et al., N. Eng. J. Med. 323:570-578. (1990), Culveret al., Science 256:1550-1552 (1992). The GlXSvNa vector is a 5865 bpvector whose map, functional features and complete DNA sequence areshown in FIGS. 2a and 2 b. FIG. 3 illustrates the procedure forconstructing the pGlPLPSvNa vector. Essentially, the pRc/CMV-PLP vectorwas digested with BstEII/HindIII and PLP encoding fragment was isolatedby gel electrophoresis. After electroelution, HindIII/NotI adapters(Stratagene, La Jolla, Calif.) were ligated into the HindIII site of theeluted fragment. A NotI digestion was performed to generate NotI ends. ANotI digest was performed on pGlXSvNa and the 5865 bp fragment wasisolated, electroeluted, and a CIAP (Calf intestine alkaline phosphatasetreatment) was performed on the fragment ends. The NotI site of theinsert was ligated into the NotI site of the vector. BstEII ends of theinsert and NotI site of the vector were Klenowed. A blunt end ligationis performed to close the vector. HB101 cells were transformed withligation mix and restriction analysis was performed to determine whichvectors contain insert and the insert orientation. The recombinantretroviruses are non-replicating and incapable of producing infectiousvirus.

[0050] 3. Retroviral Vector Supernatant

[0051] To prepare supernatant containing PLP-recombinant retrovirus, thePLP-transduced retroviral packaging cell line PA317 was grown in 4 ml ofappropriate culture medium in a T25 flask (Corning, Cambridge, Mass.).Retroviral vector supernatant is produced by harvesting the cell culturemedium when cells were 80-90% confluent, and stored in 1 ml aliquots at−70C°.

[0052] The following tests were performed on the PLP cell line and/orthe vector supernatants:

[0053] (1) The viral titer is determined using 3T3 cells. Viralpreparations with titers greater than 5×10⁴ colons forming units/ml areused.

[0054] (2) Sterility of the producer cell line and the supernatant isassured by testing for aerobic and anaerobic bacteria, fungus andmycoplasma.

[0055] The PLP-vector preparations from PA317 can be extensively testedto assure that no detectable replication competent virus is present.This is particularly relevant to the embodiment of the invention whereinthe invention is used to treat humans. Tests on both the viralsupernatant and on the transduced fibroblasts can be performed todetermine if there is replication competent virus present. The followingtests can be performed on the producer cell line and/or the viralsupernatant:

[0056] (1) The viral titer is determined using 3T3 cells. Viralpreparations with titers greater than 5×10⁴ colony forming units/ml areused.

[0057] (2) Southern blots are run on the producer cell line to detectthe partial PLP gene.

[0058] (3) PLP production by the producer cell line is measured andshould be significantly above baseline control values, as determined byELISA assay.

[0059] (4) Sterility of the producer cell line and the supernatant isassured by testing for aerobic and anaerobic bacteria, fungus andmycoplasma.

[0060] (5) Viral testing is performed including: MAP test, LCM virus,thymic agent, S+L-assay for ecotropic virus, S+L assay for xenotropicvirus, S+L-assay for amphotropic virus and 3T3 amplification.

[0061] (6) Electron microscopy is performed to assure the absence ofadventitious agents.

[0062] Following the introduction of the gene into fibroblasts, thefollowing tests are performed on the fibroblasts prior to administrationto patients.

[0063] (1) Cell viability is greater than 70′ as tested by trypan bluedye exclusion.

[0064] (2) Cytologic analysis is performed on over 200 cells prior toinfusion to assure that tumor cells are absent.

[0065] (3) Sterility is assured by testing for aerobic and anaerobicbacteria, fungus and mycoplasma.

[0066] (4) S+L-assay including 3T3 amplification must be negative.

[0067] (5) PCR assay for the absence of 4070A envelope gene must benegative.

[0068] (6) Reverse transcriptase assay must be negative.

[0069] (7) Southern blots run on the transduced fibroblasts to assurethat intact provirus is present.

[0070] (8) PLP protein assay to assure the production of PLP protein.

[0071] 4. Transfection of Fibroblasts

[0072] Prior to the transfection of the SJL fibroblasts, highly purifiedPLP-pRc/CMV vector was isolated from the transformed AG1 cells. Largescale purification was performed by using a commercially available kitand CsCl gradient banding. Initial purification was accomplished using aWizard Megaprep Kit (Promega, Madison, Wis.). A 1000 ml culture oftransformed AG1 cells, grown overnight in LB/Amp at 37° C., was pelletedand the plasmid DNA isolated according to the Manufacturer'sinstructions. The isolated DNA, which was suspended in 3 ml of TE buffer(10 mM Tris-HCl, pH 7.4, and 1 mM disodium EDTA, pH, 8.0) was furtherprocessed by CsCl gradient banding. A modified CsCl banding of the DNAwas performed based on procedures found in “Current Protocols inMolecular Biology, Vol 1” (Greene Publishing Associates andWiley-Interscience).

[0073] After the DNA band was extracted from the ultracentrifuge tubes,ethidium bromide was removed from the sample by washing it with 3volumes of SSC saturated isopropanol. The wash was repeated until theaqueous layer appeared clear. CsCl was removed by precipitation. 2volumes of 0.2M NaCl/TE and 2 volumes of 100% ethanol (relative to thecombined total volume of DNA solution and 0.2M NaCl/TE) were added tothe sample, mixed and placed on ice for 10 minutes. The precipitated DNAwas pelleted by centrifugation at 10000×g for 10 minutes at 4° C. Thepellet was washed with cold 70% ethanol, recentrifuged at 10000×g for 10minutes at 4° C., and dried under vacuum (Speed vac evaporator; SavantInc.). The purified DNA was resuspended with double-distilled sterilewater and utilized in the transfection process.

[0074] Test SJL fibroblasts were transfected using LipofectAMINE Reagent(Life Technologies Inc./Gibco BRL) according to the manufacturer'sinstructions. Control SJL fibroblasts underwent the same procedurewithout the presence of a DNA construct. 3 μg of CsCl purifiedPLP-pRc/CMV plasmid and 25 μl of Lipofectamine were used fortransfection. Approximately 3×10⁵ SJL cells, seeded overnight into 25cm² culture flasks (Corning Costar Corp., Cambridge, Mass.) and grown at37° with 5% CO₂ in 5 ml of DMEM culture medium (Dulbecco's ModifiedEagle's Medium (Irvine Scientific, Santa Ana, Calif.), supplemented with5% glutamine, 10% Fetal Calf Serum, 25 Units/ml of penicillin G sodium,and 25 μg/ml of streptomycin sulfate, were washed with 3 ml serum freeHL-1 medium (Hycor Biomedical Inc., Irvine, Calif.). After theDNA/lipofectamine complexes were incubated with cells for 6 hours at 37°with 5% CO₂, 1 ml of DMEM was added to the flasks. The flasks wereincubated overnight at 370 with 5% CO₂. The medium was replaced with 5ml of fresh DMEM the next morning. 36 hours after the end of thetransfection period, the medium was replaced with 5 ml of DMEMcontaining 900 kg of G418 (Life Technologies Inc./Gibco BRL)/ml ofmedium. The test cells were grown in the presence of 900 kg of G418 ofmedium until all the control cells had died; and no more cell deathcould be observed in the test sample flask. The G418 concentration wasthen reduced to 600 μg/ml of culture medium for duration of cellculturing procedures.

[0075] 5. Transduction of Fibroblasts

[0076] Retroviral constructs containing a neo-selectable marker togetherwith either the PLP gene or the Lac-z gene were used to transducefibroblasts. Transduction with the retrovirus was performed on healthycells (90% viable, as determined by trypan blue staining). 2×10⁶ cellswere plated in 0.5 ml DMEM-10 media (DMEM media supplemented with 109′fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin G, 50 mg/mlstreptomycin in one well of a 24-well plate (Falcon, Franklin Lakes,N.J.) Cells were placed in the incubator and allowed to settle (37°, 5%CO₂). After cells had settled, 1 ml of retroviral supernatant andpolybrene (Sigma, St. Louis, Mo.) (final concentration 10 μg/ml) wasadded to the well. Cells were incubated as above for 2.5 hours withoutshaking. After 2.5 hours, cells were transferred to a T25 flask andDMEM-10 media was added to a total volume of 8 ml. Selection media(culture media comprising DMEM-10 supplemented with 900 μg/ml G418(Gibco, Grand Island, N.Y.) was added on the third day aftertransduction. The G418 concentration was then reduced to 600 μg/ml ofculture medium for the duration of cell culturing procedures.

[0077] 6. mRNA Expression Analysis

[0078] mRNA isolation was performed using aseptic techniques, RNAse freesupplies, and DEPC (Diethylpyrocarbonate) treated solutions. 4×10⁶experimental and control SJL cells were washed twice with coldPhosphate-buffered saline, resuspended in 200 μl cell lysis mix (10 mMTRIS pH 7.5, 0.15M NaCl, 1.5 mM MgCl₂, 0.65% NP 40), vortexed, andcentrifuged at 17000×g at 40 for 5 minutes. The supernatant wastransferred to a tube containing 200 μl of urea mix (7M urea, 1% SDS,0.35M NaCl, 10 mM EDTA, and 10 mM Tris-HCL, pH 7.5) and 400 μl ofphenol:chloroform;isoamyl alcohol (25:24:1). The solution was vortexedand centrifuged for 1 minute at 17000×g. This procedure was repeatedtwice using the aqueous layer and then transferred to a tube containing400 μl of phenol and washed as before. The aqueous layer was transferredagain to another tube, and precipitated with 1 ml of 100% ethanolovernight at −20° C. The precipitated RNA was washed with 1 ml 70%ethanol. After the ethanol was discarded, the pellet was dried undervacuum lag of the RNA was used for RT-PCR analysis.

[0079] RT-PCR was performed using a commercially available kit, GeneAmpRNA PCR Kit (Perkin Elmer/ABI) according to the Manufacturer'sinstructions. The following primers were used to amplify the cDNA:5′-GCGACTACAAGACCACCATCT-3′ and 5′-TAAGGCTAGCATAGGTGATG-3′. The PCRproducts were electrophoresed on a 1.5% agarose (SeaKem GTG; FMC)/s, gelwith 1 μl of 10 mg/ml of ethidium bromide/ml of agarose solution. Thegel was electrophoresed using TAE buffer at a constant 40 mA.Electrophoresis was continued until the molecular weight marker bandshad separated adequately enough, to verify the PCR products' approximatemolecular size. The DNA band of interest was then excised and gelpurified, using the commercially available MERmaid Kit (Bio 101, Vista,Calif.), according to the Manufacturer's instructions. The purified DNAwas then sequenced by automated Fluorescent DNA sequencing procedures(Perkin Elmer/ABI, Foster City, Calif.).

[0080]FIG. 4 is an agarose gel showing PLP-specific RT-PCR products. Thedata illustrates that mRNA is present in both PLP-transduced andPLP-transfected cells. The correlation between mRNA and secreted proteinremains to be determined since peptide concentration does notnecessarily correspond to the level of mRNA.

[0081] 7. Protein Expression Analysis

[0082] The in vitro qualitative expression of the proteins encoded bythe PLP gene was detected immunologically by ELISA. Undilutedsupernatants from cultures of fibroblasts transduced with the PLP genewere tested. Wells of 96 microtiter plate were coated with thesupernatants. Primary anti-PLP-antibody 4E10 139-151, from Dr M. Lees(Harvard), is specific for PLP 139-151 and was added to wells asundiluted hybridoma supernatant followed by horseradish peroxidase(HRP)-conjugated goat anti-mouse secondary antibody in a concentrationof 1:500. The plate was developed and analyzed at 490 nm on a microplatereader. FIG. 5 illustrates the results of ELISA assays on transducedfibroblast supernatants. Samples 1 and 2 were PLP (amino acids 139-151)and HIV gp120 peptides used at a concentration of 5 ug/ml. Thisexperiment illustrates that the transduced PLP-transduced fibroblasts doproduce and secrete the partial PLP protein.

EXAMPLE 3

[0083] In Vivo Effects of the PLP Protein

[0084] Critical to the success of this invention in the embodiment ofthis example is the ability to deliver genetically manipulatedfibroblasts to patients so that the cells survive in sufficient numbersand for long periods of time, in order that continuous secreted antigenmay be provided to the patient.

[0085] To assess the fate of transplanted transduced fibroblasts, SJLfibroblasts transduced with retrovirus encoding B-galactosidase wereinjected subcutaneously between the shoulders of SJL mice. All mice werefemale mice of the SJL strain between 6-8 weeks old and were obtainedfrom Jackson Labs. Animals were housed and maintained according to NIHguidelines (National Research Council, 1986). These fibroblasts survivedin large numbers after 60 days. Fibroblasts injected into the footpad orintramuscularly could not be detected at eight days.

[0086] 1. In Vivo Fate B-Gal Transduced Cells

[0087] The activity of the B-Galactosidase marker was evaluated usingtwo groups of eight normal mice. Two mice were injected subcutaneouslyon the back, two mice were injected intramuscularly and two mice wereinjected in the footpad with Lac-Z transduced cells. One animal wasinjected with fibroblasts transduced with neo-marker only, and the lastmouse was injected with untransduced fibroblasts. After harvesting andwashing, the different cell lineages were suspended in a concentrationof 10⁷ cells in 0.2 ml of Hank's PBS and slowly injected using a 25gauge needle at different sites Animals were sacrificed at 10 and 15days post treatment and injection sites were submitted to histochemicalstudy. Pieces of tissue were fixed in 4% paraformaldehyde for one hour,washed in PBS three times and then kept in 8.4% acrylamide solutionovernight. The next morning tissues were embedded in acrylamide whichafter hardening were cut and frozen. The frozen sections were done in 10um by cryostat and stained with 1 ml of5-Bromo-4-chloro-3-indolyl-B-d-galactopyranoside (X-Gal) in PBS. TheX-Gal was dissolved in DMSO at 40 mg/ml and then added to the reactionmixture. Incubation was for 14-18 h at 37°. FIG. 6 illustrates B-Galexpression in transduced fibroblasts 60 days in vivo. There was noevidence of an inflammatory response, suggesting that the retrovirusused to transduce syngeneic fibroblasts, does not evoke an immuneresponse or rejection process.,

[0088] 2. Effect of PLP in Normal SJL Mice

[0089] Another important aspect of this invention in the embodiment ofthis example is determining whether transduced fibroblasts secreting PLPactually produce EAE in normal animals. To test this, 10′ P12-secretingSJL fibroblasts were injected into 12 normal SJL mice. Six animals hadfibroblasts placed subcutaneously and six animals had fibroblastsinjected intraperitoneally. Animals were sacrificed at day 16 and showedno evidence of inflammatory disease or EAE. FIG. 7 illustrates theclinical scoring system for chronic EAE. Y-A Lu et al., Mol. Immunol.28:623-630 (1991), J. Williamson et al., J. Neuroimmunol. 32:199-207(1991). In the EAE model for multiple sclerosis, using spinal cordhomogenates plus adjuvant, inflammation in the CNS can be seen by day14. In this study, normal animals injected with PLP-secreting SJLfibroblasts did not show any signs of clinical disease even at day 60.In addition, the animals did not show any histologic evidence ofinflammation in the CNS at day 60. FIG. 8 illustrates the histologicalscoring system for EAE. J. Governman et al., Cell 72:551-560 (1993).,

[0090] 3. Clinical and Histological Assessment of Acute EAE Mice TreatedWith Retrovirus Transduced Fibroblasts.

[0091] Six week SJL mice were infected with mouse spinal cord homogenate(MSCH) in complete Freund's Adjuvant (CFA) and with MSCH in incompleteFreund's Adjuvant IFA, seven days later. J. Immunol. 144:909-915 (1990).The initial EAE attack was observed on days 14-18, with full recovery by21. Ninety-five percent of animals showed clinical evidence of an acuteattack and these were given either 10⁷ PLP secreting SJL fibroblasts orcontrol fibroblasts on day 21. Animals not showing clinical disease wereeliminated from the experiment. FIG. 9 illustrates the clinicalassessment of EAE mice treated with retrovirus transduced fibroblasts.Animals receiving the PLP secreting fibroblasts had a marked reductionof clinical signs and had dramatic reduction in inflammatory cells,particularly in the brain.

[0092]FIG. 10a illustrates the pathologic assessment of brain and spinalcord of SJL mice treated with retrovirus transduced fibroblasts. FIG.10b is a summary of the pathologic assessment of brain and spinal cordfrom days 55-60 and 90-95. Histological assessment of EAE Grades inBrain and Spinal Cord were performed following the preparation ofhematoxylin and eosin stained sections.

[0093] 4. Clinical and Histological Assessment of Chronic EAE MiceTreated With Retrovirus Transduced Fibroblasts.

[0094] 150 mice were inoculated with MSCH in CFA. A second immunizationwas given 7 days later. A. M. Brown and D. E. McFarlin, LaboratoryInvest. 45:278-284 (1981). On day +14 to 16, 113 animals developedclinical disease lasting 3-4 days. These positive animals were separatedfor subsequent experiments and had their first relapse on day +55 to 60,with 100 animals becoming sick. These were again separated and on day+137, 67 had a relapse. Eight days after relapse, animals were eachtransplanted with 10⁷ fibroblasts and then sacrificed 18 to 23 dayslater. Four different types of fibroblasts were used, those transducedwith retrovirus encoding PLP, encoding B-galactosidase and encodingneo-selectable marker as well as untransduced cells. FIG. 11 shows thehistology of SJL mice with chronic EAE treated with retrovirustransduced fibroblasts. There were no animals receiving PLP secretingfibroblasts with 2+ to 3+ inflammation.

[0095] 5. Peripheral Immune Status of Treated Mice v. Control EAE Mice.

[0096] Spleen cells from our EAE control mice and from four EAE micewhich had been treated with fibroblasts expressing the PLP protein wereused in proliferation assays, in which they were incubated with 40 μMPLP peptide 139-151 or 40 μM HIV gp120 peptide 308-322 for 4 days andthen pulsed with ³H-thymidine for 24 hours.

[0097] Briefly, animals were sacrificed by CO₂ asphyxiation. Spleencells were dispersed to single cell suspensions in RPMI 1640 by passingthrough a size 60 mesh, and washed once before being cultured (8×10⁵ perwell) in 0.2 ml of HL-1 medium (Hycor Biomedical, Irvine, Calif.),supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 μgstreptomycin either alone or with 40 μM of peptide in 96-well tissueculture plates for 4 days at 37° C. with 5* CO₂. PLP peptide 140-151 andMBP peptide 89-101 were used for antigen-specific proliferation whileHIV gp120 peptide 308-322 was used as negative control. Where indicated,some wells also contained 10 U/ml of recombinant mouse IL-2 (BoehringerMannheim, Indianapolis, Ind.) During the last 18-24 h of culture, eachwell was pulsed with 1 μCi of ³H-thymidine (ICN, Irvine, Calif.),harvested onto ‘Xtal Scint’ glass fiber filters (Beckman, Fullerton,Calif.) and counted using a Beckman LSGOOO Scintillation counter.Thymidine incorporation values (experimental counts perminute-background counts per minute) were calculated and represent meansof triplicate cultures±standard deviation.

[0098] The results are shown in FIG. 12 and suggest that PLP specificproliferative responses are reduced significantly in EAE mice which havereceived PLP expressing fibroblasts.

[0099]FIG. 13 illustrates the same experiment as in FIG. 12 but with theaddition of mouse IL-2 (10 U/ml) for 5 days. These results illustratethat the mechanism by which the PLP specific proliferative responses arereduced significantly may suggest the possibility of deletion of T cellsrather than anergy because these lymphocytes do not respond to IL-2.

[0100] Although the mechanism by which the present invention acts torestore tolerance in individuals suffering from T-cell mediatedautoimmune disease is not entirely understood, the benefits of thetreatment are clearly advantageous over alternative treatments. Themethod is a genetic approach to immunospecifically silence pathogenicT-cell responses and does not down-regulate the entire immune system. Inthe case where an individual with a T-cell mediated autoimmune diseaseexhibits pathogenic T-cells of multiple specificities, the invention mayeasily be adapted to target those specificities. For example, DNAencoding multiple self-antigenic epitopes may be introduced into thepatient's cells. The invention is also advantageous in that the reagentscan easily be made or obtained in sufficient quantity to carry out theinvention.

[0101] The present invention is not to be limited in scope by theexemplified embodiments disclosed herein which are intended asillustrations of single aspects of the invention, and clones, DNA oramino acid sequences which are functionally equivalent are within thescope of the invention. Various modifications of the invention, inaddition to those shown and described herein, will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0102] Various publications are cited herein that are herebyincorporated by reference in their entireties.

1 1 1 5856 DNA Artificial Sal/hind drag of pSVNA into Sal/Hind cut G1 1tttgaaagac cccacccgta ggtggcaagc tagcttaagt aacgccactt tgcaaggcat 60ggaaaaatac ataactgaga ataggaaagt tcagatcaag gtcaggaaca aagaaacagc 120tgaataccaa acaggatatc tgtggtaagc ggttcctgcc ccggctcagg gccaagaaca 180gatgagacag ctgagtgatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg 240ctcggggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagtgaa 300tcatcagatg tttccagggt gccccaagga cctgaaaatg accctgtacc ttatttgaac 360taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc cgctctccga gctcaataaa 420agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac 480ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg 540ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt 600ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc 660agcaacctat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg 720tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt 780ctgaacaccc ggccgcaacc cagggagacg tcccagggac tttgggggcc gtttttgtgg 840cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt 900aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa 960ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt ctgactgtgt 1020ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt ttgaccttag 1080gtcactggaa agatgtcgag cggatcgctc acaaccagtc ggtagatgtc aagaagagac 1140gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg ccgcgagacg 1200gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca cctggcccgc 1260atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct tttgaccccc 1320ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct ccatccgccc 1380cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt tatccagccc 1440tcactccttc tctaggcgcc ggaattcgcg gccgctacgt agtcgactcg ctgtggaatg 1500tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 1560tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 1620gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 1680tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 1740ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag 1800gcttttttgg aggcctaggc ttttgcaaaa agctcgaaga tcaattccga tctgatcaag 1860agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 1920ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 1980atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 2040tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 2100cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 2160tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 2220tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 2280tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 2340tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 2400ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 2460tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 2520gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 2580gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 2640gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgtcga 2700gaagcttggg cccatcgata aaataaaaga ttttatttag tctccagaaa aaggggggaa 2760tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 2820ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag 2880ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 2940gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc 3000ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag 3060agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg 3120aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat 3180aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg 3240tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct 3300tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct 3360cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg 3420gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga 3480cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag 3540cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt 3600atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg 3660tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc 3720gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 3780ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 3840aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 3900ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 3960aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 4020gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 4080tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 4140tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 4200gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 4260cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 4320cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 4380agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 4440caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 4500ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 4560aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 4620tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 4680agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 4740gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 4800accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 4860tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 4920tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc 4980acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 5040atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 5100aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 5160tctcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 5220agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc 5280gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 5340ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 5400atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 5460tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 5520tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 5580tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 5640cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 5700ctttcgtctt caagaattca taccagatca ccgaaaactg tcctccaaat gtgtccccct 5760cacactccca aattcgcggg cttctgctct tagaccactc taccctattc cccacactca 5820ccggagccaa agccgcggcc cttccgtttc tttgct 5856

What is claimed is:
 1. A method of treating a patient for a T-cellmediated autoimmune disease comprising: introducing a cell comprising apolynucleotide encoding an antigenic amino acid sequence into a patient,the cell expressing in the patient a therapeutically effective amount ofthe antigenic amino acid sequence to induce T-cell tolerance to theexpressed antigenic amino acid sequence.
 2. The method of claim 1wherein the patient is human.
 3. The method of claim 2 wherein the cellis a fibroblast.
 4. The method of claim 3 wherein the fibroblast ishistocompatible with the patient.
 5. The method of claim 1 wherein thecell is autologous.
 6. The method of claim 1 wherein the cell isheterologous.
 7. The method of claim 1 wherein the antigenic amino acidsequence is from a nervous system protein.
 8. The method of claim 2wherein the disease is selected from the group consisting of multiplesclerosis, rheumatoid arthritis, systemic lupus erythematosus,psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease,myasthenia gravis, and chronic inflammatory demyelinating polyneuropathy(CIDP).
 9. The method of claim 8 wherein the disease is multiplesclerosis.
 10. The method of claim 1, wherein the expressed antigenicamino acid further comprises a hydrophobic leader sequence, thehydrophobic leader sequence enabling the antigenic amino acid to besynthesized in an endoplasmic reticulum for later constitutivesecretion.
 11. The method of claim 1, wherein the polynucleotide farthercomprises a Kozak box, the Kozak box permitting efficient translation ofan mRNA transcribed from the polynucleotide.
 12. The method of claim 1,wherein the antigenic amino acid sequence comprises an encephalogenicamino acid epitope.
 13. The method of claim 12, wherein theencephalogenic amino acid epitope is selected from the group consistingof the encephalogenic epitopes of myelin basic protein,myelin-oligodendrocyte glycoprotein, and proteolipid protein.
 14. Themethod of claim 13 wherein the disease is multiple sclerosis.
 15. Themethod of claim 1, wherein a recombinant vector is used to transfect thepolynucleotide into the cell.
 16. The method of claim 15, wherein thevector is a viral vector.
 17. The method of claim 16, wherein the viralvector is a retroviral vector.
 18. The method of claim 15, wherein thetransfection is in vitro.
 19. The method of claim 15, wherein thetransfection is in vivo.